Rapid detection of ACE inhibitory activity by colloidal gold method and preparation of test paper

Abstract

The invention relates to the technical field of activity detection, and particularly discloses a colloidal gold method for rapidly detecting ACE inhibitory activity and a test paper preparation method. The test paper comprises a sample pad, a gold-labeled pad, a nitrocellulose membrane and a water absorption pad, angiotensin converting enzyme labeled by colloidal gold is loaded on the gold-labeled pad, and a detection line coated with an angiotensin II antibody and a quality control line coated with an anti-ACE antibody are arranged on the nitrocellulose membrane. During detection, an ACE inhibitor in a sample can inhibit enzyme activity and influence aggregation of colloidal gold on the detection line, so that rapid judgment is realized through color comparison of the detection line and the quality control line. The invention also provides a preparation method and a detection method of the test paper, and the test paper has the advantages of simplicity and rapidness in operation, no need of large instruments, low cost, suitability for field detection and the like, and can be widely applied to drug screening, food detection and clinical sample preliminary screening.

Description

Technical Field

[0001] This invention relates to the field of activity detection technology, and in particular to the rapid detection of ACE inhibitory activity using colloidal gold method and the preparation of test strips. Background Technology

[0002] Angiotensin-converting enzyme inhibitors (ACEIs) are first-line core drugs used clinically to treat cardiovascular diseases such as hypertension and heart failure. Their target—angiotensin-converting enzyme (ACE)—is one of the key enzymes in the regulation of blood pressure in the human body. ACE is a zinc-containing dipeptide carboxypeptidase, widely found in human endothelial cells, epithelial cells, and plasma. It is the core catalytic enzyme of the renin-angiotensin system (RAS), primarily performing the following two key physiological functions: Catalyzing the conversion of angiotensin I (AngI) to angiotensin II (AngII): AngII is a potent vasoconstrictor and can promote aldosterone secretion and increase sodium and water retention, both leading to elevated blood pressure. Degrading bradykinin: Bradykinin is a peptide with vasodilatory effects. Therefore, ACE operates through a dual mechanism of "generating pressor substances (AngII)" and "eliminating antihypertensive substances (bradykinin)." If a substance can inhibit ACE activity, it means it can: reduce the production of AngII, thereby directly dilating blood vessels and reducing peripheral resistance; and slow down the degradation of bradykinin, indirectly enhancing its vasodilatory effect. The synergistic effect of these two actions makes ACE inhibitors highly effective and reliable antihypertensive drugs (such as captopril and enalapril).

[0003] Currently, the mainstream methods for detecting ACE inhibitory activity in laboratories mainly include: (1) High performance liquid chromatography (HPLC) is used to directly and quantitatively determine the amount of AngII produced by ACE catalytic reaction. After the reaction is terminated, AngI and AngII are separated by HPLC and the peak area of ​​AngII is quantified by detector (such as UV or mass spectrometry) to calculate the inhibition rate.

[0004] (2) Spectrophotometry / fluorescence spectrophotometry uses artificially synthesized chromogenic or fluorescent substrates (most commonly hippuryl-histyl-leucine, HHL). ACE hydrolyzes HHL to release hippuric acid and histyl-leucine (HL), the latter of which reacts with derivatizing reagents (such as phthalaldehyde) to generate a strongly fluorescent product. After the enzymatic reaction, a derivatizing reagent is added, and the fluorescence intensity is measured using a fluorescence spectrophotometer to indirectly calculate enzyme activity. This method can also be used to directly detect the absorbance of hippuric acid using a UV-Vis spectrophotometer.

[0005] However, both high-performance liquid chromatography and spectrophotometry require incubation, reaction, sample injection and analysis, involving multi-step liquid transfer, pH adjustment, reaction control and special equipment. Therefore, there is an urgent need in this field for a new technology for detecting ACE inhibitory activity that can overcome the above-mentioned shortcomings, achieve rapid, simple, low-cost detection without large instruments and suitable for on-site detection. Summary of the Invention

[0006] This application provides a method for rapid detection of ACE inhibitory activity using colloidal gold and the preparation of test strips, which solves the problem of complex processes and equipment required in the prior art.

[0007] This application provides a test strip for rapid detection of ACE inhibitory activity using a colloidal gold method, comprising: The sample pad, gold label pad, nitrocellulose membrane, and absorbent pad are sequentially attached to the base plate; The gold-labeled pad is loaded with colloidal gold-labeled angiotensin-converting enzyme; The nitrocellulose membrane is provided with a detection line and a control line. The detection line is coated with angiotensin II antibody, and the control line is coated with anti-ACE antibody.

[0008] Furthermore, the method for preparing the colloidal gold-labeled angiotensin-converting enzyme includes: Adjust the pH of the colloidal gold solution to 7.4-7.8, add angiotensin-converting enzyme, react at room temperature for 10-30 minutes, add blocking agent to block, centrifuge, and resuspend in buffer containing stabilizer.

[0009] Furthermore, the method for preparing the colloidal gold solution includes: Add chloroauric acid solution to ultrapure water, heat to boiling, quickly add sodium citrate solution, and continue heating until the solution turns wine red or purple-red. After cooling, make up to volume.

[0010] Furthermore, the amount of sodium citrate solution added is 1.5-2.5 times the volume of chloroauric acid solution.

[0011] Furthermore, the coating concentration of angiotensin II antibody on the detection line is 0.4-0.8 mg / mL, and the coating concentration of anti-ACE antibody on the quality control line is 1.0 mg / mL.

[0012] Furthermore, the sample pad is pretreated with a buffer solution containing surfactants and stabilizers and then dried; the gold label pad is pretreated with a buffer solution containing surfactants and sugars and then dried.

[0013] The preparation method of the above test strip includes the following steps: (1) Preparation of colloidal gold solution; (2) Preparation of colloidal gold-labeled angiotensin-converting enzyme; (3) Angiotensin II antibody and anti-ACE antibody were respectively sprayed onto nitrocellulose membrane to form detection lines and control lines; (4) The pretreated sample pad, the gold label pad loaded with colloidal gold labeled enzyme, the nitrocellulose membrane and the absorbent pad are sequentially attached to the base plate.

[0014] The method for detecting ACE inhibitory activity using the above-mentioned test strips includes: Add the sample to be tested to the sample pad, and observe the color development of the test line and the control line after reacting for 10-20 minutes. If the control line shows color and the test line shows a deep color, the result is considered negative. If the control line shows color and the test line shows light color or no color, it is considered positive. If the control line does not show color, it is considered invalid.

[0015] One or more technical solutions provided in the embodiments of this application have at least the following technical effects or advantages: Existing technologies rely on endpoint methods or kinetic monitoring, requiring sufficient reaction time to achieve a detectable product amount. Furthermore, the instrumental analysis steps themselves are cumbersome and time-consuming. The capture and quantification of detection signals (chromatographic peaks, absorbance, fluorescence values) depend entirely on these sophisticated optical or separation instruments. The colloidal gold method for rapid detection of ACE inhibitory activity and the test strip preparation technology provided in this application embodiment achieve rapid and simple detection of ACE inhibitory activity, requiring no complex instruments or professional operators throughout the process, and is suitable for on-site and rapid screening scenarios. Through a cascade reaction of colloidal gold-labeled enzyme and antibody capture on a nitrocellulose membrane, enzyme inhibitory activity is converted into a straightforward color signal, with a detection time of only 10-20 minutes. The test strip preparation process is mature and inexpensive; performance can be optimized by adjusting parameters such as colloidal gold particle size, enzyme labeling amount, and antibody concentration to adapt to different sensitivity and cost requirements. Compared with traditional chromatography and spectrophotometry, this method eliminates complex steps such as incubation, pipetting, derivatization, and instrumental analysis, significantly improving detection efficiency and reducing technical barriers and operating costs. Detailed Implementation

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention; the term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0017] Example 1: This example provides a rapid detection method for ACE inhibitory activity using colloidal gold, the detection steps of which include: S1. Preparation of colloidal gold solution: Acid tank cleaning: Immerse all glassware in acid tank solution (400g K2Cr2O7 + 3L ultrapure water + 10L concentrated H2SO4 until completely dissolved) for 3 days, rinse with ultrapure water, sterilize and dry.

[0018] Synthesis: Add 1.0 mL of 1% chloroauric acid solution to 100 mL of ultrapure water, heat to boiling with magnetic stirring, quickly add 2.0 mL of 1% sodium citrate solution, continue heating and stirring until the solution turns wine red (about 10 min), stop heating, cool and make up to 100 mL, store at 4℃ protected from light; obtain colloidal gold solution.

[0019] Characterization: The solution is wine-red, and the maximum absorption peak in the ultraviolet-visible spectrum is at 520 nm.

[0020] S2. Determination of the optimal enzyme labeling amount: Take 3 mL of colloidal gold solution and adjust the pH to 8.0 with 0.2 M K2CO3. Add 100 μL of the pH-adjusted colloidal gold solution to each well of the microplate, and add 0, 5, 10, 15, 20, and 25 μL of ACE enzyme (0.1 U / mL) respectively. Let it stand at room temperature for 10 min. Add 10 μL of 10% NaCl solution to each well, mix well, and let it stand for 30 min to observe the color. The minimum enzyme amount that keeps the color red and does not turn blue is 15 μL (i.e., 1.5 mU of ACE enzyme per 100 μL of colloidal gold).

[0021] S3. Optimization of colloidal gold labeling enzyme conditions (pH): Take 6 aliquots of 1 mL colloidal gold solution and adjust the pH to 6.0, 6.4, 6.8, 7.2, 7.6, and 8.0 respectively; add 5 μL of ACE enzyme (0.1 U / mL) to each tube, react at room temperature for 10 min, then add 100 μL of 10% NaCl solution and let stand for 30 min; the labeling is most stable at pH 7.6 (red color is maintained), so pH 7.6 is selected as the optimal labeling pH.

[0022] S4. Preparation of colloidal gold labeled enzyme: Take 10 mL of colloidal gold solution and adjust the pH to 7.6. Add ACE enzyme according to the optimal labeling amount (15 μL of 0.1 U / mL enzyme per 100 μL of colloidal gold). Stir slowly at room temperature for 30 min. Add 100 μL of 10% BSA solution to block for 1 h. Centrifuge at 8500 rpm for 30 min at 4℃. Discard the supernatant. Resuspend the precipitate in 100 μL of resuspension buffer (0.01 MPBS containing 0.1% BSA, pH 7.4) and store at 4℃ in the dark.

[0023] Selection of optimal coating concentration for S5 and NC membranes: C line: Anti-ACE antibody was diluted to 1.0 mg / mL with 0.01 M PBS (pH 7.4) and sprayed onto the NC membrane as a quality control line.

[0024] T line: Ang II antibody was diluted to 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL, respectively, and sprayed onto the NC membrane as the detection line.

[0025] Using negative (without inhibitor) and positive (containing 1 μM captopril) samples for testing, the T-line concentration of 0.6 mg / mL showed the most significant contrast between positive and negative results, so this concentration was selected.

[0026] S6. Assembly of the test strip: Gold-labeled pads: Glass fiber cotton (3 mm × 8 mm) was soaked in treatment solution (0.1% Triton X-100, 1% sucrose in 0.01 M PBS, pH 7.4) and dried at 42°C for 4 h. Colloidal gold-labeled enzyme was then uniformly sprayed onto the pads and dried at 37°C for 2 h.

[0027] Sample pad: Glass fiber cotton (3 mm × 15 mm) was soaked in treatment solution (0.1% Tween 20, 0.5% BSA in 0.01 M PBS, pH 7.4) and dried at 42°C for 4 h.

[0028] NC membrane (3 mm × 25 mm): T line (0.6 mg / mL Ang II antibody) was sprayed 8 mm from one end, and C line (1.0 mg / mL anti-ACE antibody) was sprayed at 5 mm intervals. The membrane was then dried at 37°C for 4 h.

[0029] Assembly: On the PVC base plate (3 mm wide), attach the sample pad, gold label pad (2 mm overlap with the sample pad), NC film (2 mm overlap with the gold label pad), and absorbent pad (2 mm overlap with the NC film) in sequence, then insert into the casing and seal for storage.

[0030] S7. Optimization of reaction time: Using the same positive sample (1 μM captopril), observations were made at 10, 15, and 20 min after sample addition. The T / C line color stabilized at 15 min, therefore the optimal reaction time was 15 min.

[0031] Test strip performance testing: Sensitivity: Captopril standard gradient test (0, 0.5, 1, 2, 4, 8 µg / L), the lowest detection limit is 1 µg / L (T line becomes significantly lighter).

[0032] Interpretation: C line is bright and T line is dark → Negative; C line is bright and T line is light → Positive; C line is not bright → Invalid.

[0033] Example 2 (Increasing the particle size of colloidal gold): The only difference from Example 1 is that the amount of sodium citrate used in the preparation of colloidal gold is increased, resulting in colloidal gold particles with a slightly larger particle size (absorption peak red shift), and the labeling conditions are optimized accordingly. S1. Preparation of colloidal gold solution: Acid tank cleaning is the same as in Example 1.

[0034] Synthesis: 100 mL ultrapure water + 1.0 mL 1% chloroauric acid, heat to boiling, then quickly add 2.5 mL 1% sodium citrate solution, continue heating until the solution turns purple-red, cool and dilute to volume.

[0035] Characterization: The solution is purple-red, with the maximum absorption peak at 530 nm.

[0036] S2. Determination of the optimal enzyme labeling amount: The method is the same as in Example 1. The minimum enzyme amount that keeps the red color from turning blue is 20 μL (0.1 U / mL) per 100 μL of colloidal gold.

[0037] S3. Optimization of colloidal gold labeling enzyme conditions (pH): The pH range was tested using the same method from 6.0 to 8.0, with the optimal labeling pH being 7.8. S4. Preparation of colloidal gold labeled enzyme: Take 10 mL of colloidal gold and adjust the pH to 7.8. Add 20 μL of 0.1 U / mL ACE enzyme per 100 μL of colloidal gold for labeling. The subsequent blocking, centrifugation and resuspending steps are the same as in Example 1.

[0038] S5, Selection of optimal coating concentration for NC membrane: C line remains 1.0 mg / mL anti-ACE antibody; after antibody testing of T line, 0.4 mg / mL was selected as the optimal coating concentration (because the signal is enhanced by the increase in particle size, the amount of antibody required is reduced).

[0039] S6. Assembly of test strips: Each component is processed in the same way as in Example 1, except that the T line coating concentration is 0.4 mg / mL.

[0040] S7. Optimization of reaction time: The optimal reaction time remains 15 min.

[0041] Test strip performance testing: Sensitivity: The limit of detection is 0.8 µg / L (slightly better than Example 1); the interpretation criteria are the same as in Example 1.

[0042] Example 3 (Reducing enzyme labeling amount and increasing antibody concentration): The difference from Example 1 is that the amount of ACE enzyme labeling is reduced while the concentration of T-line antibody coating is increased in order to explore a more economical process.

[0043] S1. Preparation of colloidal gold solution: Same as in Example 1 (absorption peak 520 nm).

[0044] S2. Determination of the optimal enzyme labeling amount: Screening revealed that the enzyme amount could be reduced to 10 μL (0.1 U / mL) per 100 μL of colloidal gold. After adding NaCl, it still maintained a red color (slightly less stable). This low enzyme amount was selected to save costs.

[0045] S3. Optimization of colloidal gold labeling enzyme conditions (pH): The optimal labeling pH is 7.4.

[0046] S4. Preparation of colloidal gold labeled enzyme: Take 10 mL of colloidal gold and adjust the pH to 7.4. Add 10 μL of 0.1 U / mL ACE enzyme per 100 μL of colloidal gold for labeling. The subsequent steps are the same as in Example 1.

[0047] S5. Selection of optimal coating concentration for NC membrane: C line remains 1.0 mg / mL anti-ACE antibody; due to the decrease in enzyme labeling and weakened signal, increasing the T line antibody concentration to 0.8 mg / mL can obtain the best positive and negative contrast.

[0048] S6. Assembly of the test strip: The T-line coating concentration is 0.8 mg / mL, and the rest is the same as in Example 1.

[0049] S7. Optimization of reaction time: The optimal reaction time is 20 min (the reaction is slightly slower when the amount of enzyme is small).

[0050] Test strip performance testing: Sensitivity: The lowest detection limit is 2 µg / L (sensitivity is slightly lower than that in Example 1); the interpretation criteria are the same as in Example 1.

[0051] Example 4: This example is an improvement on Examples 1-3. After completing the routine test using the test strip, the test strip is gently heated to accelerate solvent evaporation, making the color signal more stable and clear. This verifies the test results and allows for secondary confirmation of weakly positive or borderline samples, improving the reliability and accuracy of the test.

[0052] After 15 minutes of routine testing, observe and record the color development of the T / C line under natural light, and interpret the positive or negative result according to the standard. Then, heat the test strip horizontally and uniformly heat the NC membrane area (temperature 40℃±2℃) for 3 minutes. After heating, wait for the test strip to cool to room temperature (about 1 minute), and observe and record the color development of the T / C line again.

[0053] Verification and secondary confirmation criteria: Scenario A (Verification passed, result confirmed): After heating, the color of line C remains or slightly darkens, indicating that the test strip is effective.

[0054] The T-line color change trend is consistent with expectations: for initially negative samples, the T-line color should remain dark or slightly darker; for initially positive samples, the T-line color should remain light or unchanged.

[0055] At this point, the result after heating is used as the final report result because its signal is more stable.

[0056] Scenario B (Test strip failure warning): After heating, the C line becomes significantly lighter, disappears, or diffuses abnormally.

[0057] This phenomenon suggests that the test strip may have been stored improperly (such as being exposed to moisture or high temperatures), causing the antibody or colloidal gold complex to become inactive. Therefore, the test result is unreliable and a new test strip should be used for retesting.

[0058] Case C (weak positive / borderline sample confirmation): During routine interpretation, the color of the T-line is in a borderline state between "light" and "dark", making it difficult to judge.

[0059] After heating, the signal contrast is enhanced due to the reduction of background solvent. If the sample is a true positive, the T-line color will not change significantly or will become lighter because there is no excess colloidal gold to aggregate; if it is a false positive or negative, the residual colloidal gold may aggregate slightly and become darker due to water evaporation.

[0060] This method provides auxiliary criteria for judging fuzzy results, and when combined with positive and negative comparisons, it can improve the accuracy of interpretation.

[0061] Performance testing: The same batch of test strips (n=20) from Example 1 were subjected to both routine reading and heat-verification reading, using known concentrations of captopril standards (0, 0.2, 0.4, 0.6, 0.8, 1 µg / L). Sensitivity: After heating verification, the detection limit was stabilized at 0.8 µg / L from the usual 1 µg / L, thus reducing background interference.

[0062] Repeatability: After heating validation, the intra-batch coefficient of variation (CV) for the same concentration samples with varying shades of T-line color decreased by approximately 15%.

[0063] This embodiment achieves the following significant effects through a gentle, timed, and controllable heating verification step: Results consolidation and confirmation: Gentle heating removes residual solvent, making the specifically bound colloidal gold signal more stable and reducing interpretation errors caused by excessive ambient humidity or observation time deviation.

[0064] Quality control upgrade: The single C-line colorimetric control has been upgraded to a dual quality control of "C-line colorimetric development + stability after heating", which can more sensitively detect potential failures caused by reagent degradation.

[0065] Auxiliary Critical Value Interpretation: This feature provides a physical method for secondary interpretation of weakly positive or borderline samples that are difficult to distinguish in conventional interpretation, thus improving detection accuracy. A heating verification step is introduced, which accelerates solvent evaporation through gentle heating, enhances signal contrast, improves the accuracy of interpreting weakly positive or borderline samples, and achieves dual quality control of the test strip's effectiveness.

[0066] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A test strip for rapid detection of ACE inhibitory activity using a colloidal gold method, characterized in that, include: The sample pad, gold label pad, nitrocellulose membrane, and absorbent pad are sequentially attached to the base plate; The gold-labeled pad is loaded with colloidal gold-labeled angiotensin-converting enzyme; The nitrocellulose membrane is provided with a detection line and a control line. The detection line is coated with angiotensin II antibody, and the control line is coated with anti-ACE antibody.

2. The test strip as described in claim 1, characterized in that, The method for preparing the colloidal gold-labeled angiotensin-converting enzyme includes: Adjust the pH of the colloidal gold solution to 7.4-7.8, add angiotensin-converting enzyme, react at room temperature for 10-30 minutes, add blocking agent to block, centrifuge, and resuspend in buffer containing stabilizer.

3. The test strip as described in claim 2, characterized in that, The method for preparing the colloidal gold solution includes: Add chloroauric acid solution to ultrapure water, heat to boiling, quickly add sodium citrate solution, and continue heating until the solution turns wine red or purple-red. After cooling, make up to volume.

4. The test strip as described in claim 3, characterized in that, The amount of sodium citrate solution added is 1.5-2.5 times the volume of chloroauric acid solution.

5. The test strip as described in claim 1, characterized in that, The concentration of angiotensin II antibody on the detection line is 0.4-0.8 mg / mL, and the concentration of anti-ACE antibody on the quality control line is 1.0 mg / mL.

6. The test strip as described in claim 1, characterized in that, The sample pad was pretreated with a buffer solution containing surfactants and stabilizers and then dried; the gold label pad was pretreated with a buffer solution containing surfactants and sugars and then dried.

7. A method for preparing a test strip as described in any one of claims 1-6, characterized in that, Includes the following steps: (1) Preparation of colloidal gold solution; (2) Preparation of colloidal gold-labeled angiotensin-converting enzyme; (3) Angiotensin II antibody and anti-ACE antibody were respectively sprayed onto nitrocellulose membrane to form detection lines and control lines; (4) The pretreated sample pad, the gold label pad loaded with colloidal gold labeled enzyme, the nitrocellulose membrane and the absorbent pad are sequentially attached to the base plate.

8. A method for detecting ACE inhibitory activity using the test strip as described in any one of claims 1-6, characterized in that, include: Add the sample to be tested to the sample pad, and observe the color development of the test line and the control line after reacting for 10-20 minutes. If the control line shows color and the test line shows a deep color, the result is considered negative. If the control line shows color and the test line shows light color or no color, it is considered positive. If the control line does not show color, it is considered invalid.